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2 Feb. 27, 2014 Drug Screening : from Phenotypes to Molecular Modeling Welcome to the first Rhône-Alpes meeting promoting the chemical biology topic : «Drug Screening : from Phenotypes to Molecular Modeling» A few time ago Dr. Ronald Frank, Coordinator of the EU-PENSCREEN European project wrote : «Why investigate the biological effects of chemical substances? Chemical substances can kill bacteria (but also man!), can block the spread of viruses, hinder the growth of cancer cells, protect plant growth, and many more things. Nature has produced a virtually infinite variety of molecules in environments ranging from the depths of the oceans, to rain forests, to natural oil sources. Additionally, chemists have been continuing to develop novel, artificial compounds. This has produced an immense reservoir of potential drugs that might, for instance, prevent or treat diseases if only scientists could determine the various effects of these many substances. Across the world, scientists are seeking substances that can solve particular health challenges in a targeted way. This is taking place in private and public laboratories that have, so far, worked mainly in an isolated manner. nly joining forces and overcoming this fragmentation will allow us to learn everything we can about each substance to understand the mechanisms behind its activity and to recognize potential hazardous effects as early as possible» Chemical Biology is an evolving interdisciplinary research field that studies biological processes using chemical techniques and tools. Chemical entities are introduced into biological systems as nonnatural building blocks, tags, enzyme substrates or ligands (binders); they then selectively modify cellular target molecules to become activated, inhibited or labelled for removal (pull-down) or visualisation. ne major focus is on developing chemical substances to be used as probes that interact with defined sites on the surfaces of target cellular molecules, such as proteins, and thereby selectively modulate their biological function. When applied to cells or organisms, the consequences of this perturbation, such as proliferation, differentiation, death etc., are studied in molecular detail. Such chemical probes help scientists to reveal the exact role of cellular components in the complex network of cellular processes and responses. Simultaneously, the value of a compound as potential effective reagent and future product is made apparent. Today we are pleased to welcome in Villeurbanne, France about 80 participants from very different fields belonging to chemistry, cell biology, physical chemistry, etc. We hope that this meeting will help them in discussing their research projects and, hopefully, construct new collaboratives ideas for the best of the chemical biology development in the Rhône-Alpes Region. Jean-Marc Lancelin, Université Lyon 1, Marie-dile Fauvarques, CEA-DSV Grenoble Co-organizers Drug Screening : from Phenotypes to Molecular Modeling 2

4 Access by train or by plane: From Lyon-St Exupéry airport: Airport Lyon in less than 30 minutes : From Part-Dieu train station: Take the T1 Tramway towards IUT Feyssine and get off at INSA-Einstein. About 15 min transportation. Tickets are available from the vending machines at each tram stops. From Perrache train station: Take the Line A metro towards Laurent Bonnevay and get off at "Charpennes", then take the T1 Tramway towards IUT Feyssine and get off at "INSA-Einstein". Access from the highway: Via "Rocade Est" ring road: exit 1B then "Croix Luizet", follow "la Doua", then "Domaine Scientifique de la Doua". Via the Boulevard Laurent BNNEVAY: exit 6 "Porte de Croix Luizet", then follow direction "Campus de la Doua" (road access map at Public Transport of Lyon (TCL) All the information to travel in public transports on: Drug Screening : from Phenotypes to Molecular Modeling 4

8 Plenary Lecture 1 In-silico ligand binding assays: poses, affinities and kinetics Gianni De Fabritiis Computational Biophysics Laboratory (GRIB-IMIM),Universitat Pompeu Fabra, Barcelona Biomedical Research Park (PRBB),C/ Dr. Aiguader 88, 08003, Barcelona, Spain. Tel (0506) Fax ffice: Unit page: Lab page: Understanding kinetics and thermodynamics properties of protein-ligand interactions by computer simulations is of critical importance for biomedical research. Recently, we have been able to quantitatively reconstruct the complete binding process of several molecular systems in terms of binding poses, kinetics, affinities and pathways of binding. The methodology is based on performing high-throughput molecular dynamics simulations of free ligand binding with the aim of recovering binding poses with accuracy of less than 2 Å RMSD compared to crystal structures and associated kinetics. Furthermore we obtain secondary binding sites which can be of importance fragment based drug design approaches. We assess in this talk the current capabilities of the methodology and its accuracy and precision on a set of protein-ligand systems which demonstrate the potential for drug design. References Drug Screening : from Phenotypes to Molecular Modeling 8

10 Introduction to Drug Discovery & a Case Study of Phenotypic Screening Philippe Masson, Head of Biology, screening and Compound Management, Inventiva Pharma The first part of the presentation will focus on drug discovery concepts and challenges. Despite huge investments Pharmaceutical industry productivity is decreasing. Better understanding of the biological mechanisms and innovation is probably one key for future success. After decades of target-based screening, more biology-integrated assays like phenotypic screening could be a valuable approach. In a second part, an example of phenotypic screening to find anti-fibrotic compounds for treating chronic kidney disease (CKD) will be presented. CKD is a major public health problem mainly causes by diabetes and hypertension. A common feature of these CKD is the fibrotic status which settles down progressively over years. Using a high-content screening approach, renal fibroblast treated by TGFβ1, a major fibrotic factor, clearly induced a fibrotic response based on the increase of extracellular matrix deposition, fibroblast differentiation, and proliferation. These effects are blocked, in dose dependent manner, by an Alk5 inhibitor. 51K compounds were screened at 3 µm using this four colour assay. 47 compounds, not toxic, were founded to block, in dose dependent manner, the three fibrotic parameters all together induced by TGFβ1. 21 compounds distributed into 6 families ( 2 hits) and 26 singletons displaying IC50 values from >30µM to 0.1µM. Activity of these 47 hits was confirmed by qpcr on Fibronectin1 and Acta2 genes, indicating that the compounds do not act through post translational modification of fibronectin and αsma. In order to check the activity of compounds in a human fibroblastic context, MRC5 cells treated with TGFβ1 were incubated with the 47 hits. qpcr analyses performed on fibronectin1, acta2, collagen Iα1 and collagen IVα1, showed that 11 hits were still able to block in human cells the induction by TGFβ1 on these four genes. Interestingly some of these hits did not block the TGFβ1 binding or TGFβ receptor kinase activities. These data support the fact that a phenotypic screening can deliver innovating hits that act on conserved TGFβ1 pathways without directly involving the TGFβ receptors. In a third part, slides about Inventiva a new Partnering Research Company (PR) will be presented.

12 Using NMR and molecular dynamics as a microscope for life science. Application to the determination of protein- ligand affinity livier WALKER Institut des Sciences Analytiques, 5 rue de la Doua, Villeurbane, France Molecular interactions are of prime importance to ensure communication within cells. Capturing theses processes requires the use of a method sensitive to both structure and dynamics at an atomic level. As a suitable method, NMR can probe dynamics processes in the liquid state through observables averaged over different time scales. To fill the gap, CPU accelerated molecular dynamics (MD) can provide a valuable piece of information to interpret and decipher NMR data. Through different examples, we will see how NMR, MD and funnel metadynamics(1,2) can help to understand protein- ligand interactions and affinity. A) B) Figure 1: (Left) definition of the funnel used to explore binding modes of a small compound to the SH3 domain of STAM2, (Right) Free energy as a function of the projection on the z axis and distance from z axis of the center of mass of the ligand, where z is the axis of the funnel. Reference: 1. Limongelli, V., Bonomi, M., and Parrinello, M. (2013) Funnel metadynamics as accurate binding free- energy method. Proc Natl Acad Sci U S A 110, Harvey,M., Giupponi, G. and De Fabritiis,G. (2009) ACEMD: Accelerated molecular dynamics simulations in the microseconds timescale, J. Chem. Theory and Comput. 5, 1632

13 TAGGING LIVE CELLS WHICH EXPRESS SPECIFIC PEPTIDASE ACTIVITY WITH SLID-STATE FLURESCENCE Maxime PRST, 1,2 Laurence CANAPLE, 2 Jacques SAMARUT, 2 Jens HASSERDT 1 1 Laboratoire de Chimie, ENS de Lyon, Lyon, France 2 Institut de Génomique Fonctionnel de Lyon, ENS de Lyon, Lyon, France Detecting a specific enzyme activity has long been of great interest because it is applied in fields as diverse as histology, biotechnology or medical diagnostics. However current probes usually suffer from a lack of robustness (false positive signal), from swift degradation by photobleaching, and from poor sensitivity which makes them unsuitable for precise enzymatic activity localization. [1] To overcome the above mentioned problems, we have developed three-component fluorogenic probes which allow very precise and sensitive localization of a specific active enzyme by releasing a unique solid-state fluorophore (Figure 1). Figure 2 : Imaging Leucine Amino Figure 1 : Principle of the three-component probes Peptidase in HeLa cells (25 µm for 2 h) This phenolic fluorophore, known as ELF-97 alcohol, [2] is only fluorescent in solid state, possesses an unusually large Stokes shift, is totally photostable, and can be turned off if its phenolic proton is replaced by another group. However, this compound has not been widely used because of the instability of the chemical link between the fluorophore and the enzyme-susceptible portion of the probe. We have overcome this issue by incorporating a smart spacer ensuring complete probe stability. After catalytic cleavage, a metastable intermediate is generated that cyclizes thereby releasing the fluorophore (Figure 1). The simple four-step synthesis [3] allowed us to create a variety of fluorogenic probes incorporating several spacers, fluorophores, and enzyme-susceptible trigger units. The resulting constructs were first characterized in vitro against their specific purified enzymes, and the most promising ones tested in cellulo. Thus, we were able to tag HeLa cells expressing LecuineAminoPeptidase (Figure 2) with micrograins of fluorescent solid.[4] References: [1] E. Boonacker, C. J.F. Van Noorden J. Histochem. Cytochem. 2001, 49, 1473 [2] V.B. Paragas, J.A. Kramer, C. Fox, R.P. Haugland, V.L. Singer J. Microscopy 2002, 206, 106 [3]. Thorn-Seshold, M. Vargas-Sanchez, S. McKeon, J. Hasserodt Chem. Commun. 2012, 48, 625 [4] M. Prost, L. Canaple, J Samarut, J. Hasserodt, Submitted

15 Gscore, a Robust Cell-by-cell Score for Sensitive and Specific Hit Discovery in High Content Screening Laurent GUYN 1,2,3, Christian LAJAUNIE 4,5,6, Frédéric FER 1,2,3, Ricky BHAJUN 1,2,3, Mélissa MARY 1,2,3, Eric SULPICE 1,2,3, Guillaume PINNA 7, Anna CAMPALANS 8, J. Pablo RADICELLA 8, Stéphanie CMBE 1,2,3, Patricia BEID 1,2,3, Jean-Philippe VERT 4,5,6, Xavier GIDRL 1,2,3 1 Université Grenoble-Alpes, F Grenoble, France 2 CEA, Laboratoire BGE, irtsv, 17 rue des Martyrs, F Grenoble cedex 9, France 3 INSERM, U1038, F Grenoble cedex 9, France 4 Centre for Computational Biology - CBI, Mines ParisTech, 35 rue Saint-Honoré, Fontainebleau, F France 5 Institut Curie, 26 rue d'ulm, Paris, F France 6 INSERM, U900, Paris, F France 7 CEA, Plateforme ARN interference PArI, F Gif-sur-Yvette, France 8 CEA, Institute of Cellular and Molecular Radiobiology, F Fontenay aux Roses, France Keywords: High Content Screening (HCS), scoring, hit finding, False Positive reduction. High Content Screening (HCS) has enabled great advances both in oncology and biology. It consists in visualizing phenotypes modification of cells after perturbation. This perturbation is generally achieved either by chemical compounds or RNA interference in a highly parallel manner (in 384 well-plates for example). HCS experiments produce huge amount of data, typically tables of millions of rows and tens of columns; each row corresponding to a cell whose phenotype is characterized with different metrics (each column). After analysis, hits, which are the biggest modifiers of the cell phenotype, are extracted. A method of choice, Zscore, which averages the fluorescence for each cell modified by a given compound, has proven great efficiency when fluorescence is used to monitor the phenotype. However it faces a few drawbacks: 1. Zscore is very sensitive to artifacts (aberrant fluorescence of just one cell will affect the score) 2. Low cell number for a given treatment will strongly increase Zscore variability 3. Zscore is associated to a pvalue only when fluorescence distribution is Gaussian In situations where the consequences of perturbation are strong and overtake such artifacts, Zscore find hits with a low False Discovery Rate. However in conditions closer to the physiological reality (such as rare cells or cells from patient), one need to overcome these pitfalls. Thus, in an attempt to improve the potential of discovery of HCS, we developed a so called Gscore, which is based on the rank of the fluorescence and takes into account the number of cells in a given treatment with an appropriate model. I will show the advantages of the score compared to the widely used Zscore in various situations using virtual screen and real screening data, including a screen of gene knock-down reagents (druggable collection, targeting more than 7000 genes).

17 Treating yeast infections with new innovative chromatin targets Morgane Champleboux 1, Flore Mietton 1, Elena Ferri 4, Didier Spittler 2, Muriel Cornet 3, Charles McKenna 4, Carlo Petosa 2 and Jérôme Govin 1 1. Institute of Research in Life Sciences and Technologies, Department Large Scale Biology, 17 Rue des Martyrs, Genoble Cedex 9 2. Institut de Biologie Structural, 41, rue Jules Horowitz, Grenoble Cedex 1 3. Laboratoire TIMC-TheREx Domaine de la Merci, La Tronche 4. University of Southern California, Department of Chemistry, 3620 McClintock Avenue, Los Angeles, CA USA C. albicans is the most prevalent human fungal pathogen and is responsible for the most deaths. With only four drug classes available to treat invasive fungal infection, there is an urgent need to find new therapeutic agent, to overcome the emergence of drug-resistant strains, problems related to the toxicity and narrow activity spectrum of existing drug. Bromodomain proteins are chromatin-associated factors that regulate gene transcription and chromatin remodelling. They recognize short peptides acetylated on lysine residues, and are involved in many processes like cancer development, infection and reproduction. Recently, several efforts of academic groups and biopharmaceutical companies have led to the discovery of several potent and selective human bromodomain inhibitors, with promising outcomes in cancers and human pathologies. This project explores the functional role of bromodomain proteins in C. albicans, and their potential as therapeutic targets. We investigate their role in C. albicans biology, and develop an ambitious program to identify yeast-specific bromodomain inhibitors..

18 Title : STLC- resistant cell lines as tools to classify chemically divergent Eg5 targeting agents according to their mode of action and target specificity Isabel Garcia- Saez, Salvatore DeBonis, Rose- Laure Indorato, Françoise Lacroix, Dimitrios Skoufias Institut de Biologie Structurale, UMR5075 (CNRS- CEA- UJF). Grenoble 38027, France The microtubule based kinesin Eg5, also known as KIF11, has been recognized as a valid target for the development of new class of anti- mitotic inhibitors targeting components of the mitotic spindle with a potential cancer chemotherapeutic value (1). To date, a number of chemically distinct small molecules targeting different binding pockets of Eg5 protein are under evaluation in clinical trials with better responses achieved when hematological malignancies were targeted. ne of the Eg5 inhibitors, ARRY- 520 is set to enter late- stage clinical testing in several hundred human patients with relapsed or refractory multiple myeloma. We have been addressing the issue of specificity and resistance to Eg5 inhibitors and in particular to S- Trityl_L- Cysteine (STLC) that we have previously identified based on enzymatic screening. Based on the structure of the STLC- Eg5 complex, we have been successful in developing drug resistant cell lines as tools to classify chemically divergent Eg5 targeting agents according to their mode of action and target specificity (2,3). We have also identified Eg5 mutants encountered in a number of selected drug resistant clones of colon carcinoma tumor cells and we are carrying out structure activity studies in one of such mutant. Interestingly, this mutation, instead of being localized in the known allosteric drug- binding pocket of Eg5, is in the nucleotide- binding site. ur current biochemical data coupled with cell- based assays support the hypothesis that the mutant, in the absence of the inhibitor, is in a rigor state (high friction mode) and free of nucleotide. When the mutant is expressed in cells, heavily crosslinked MTs are formed. Strikingly, the MT bundles are released in the presence of the inhibitor. The drug resistant cell lines can therefore be used as a filter to distinguish Eg5 loop L5 binding drugs without prior structural information. Additionally, the cells can be used to analyze whether inhibitors of Eg5 are specific to this potential drug target or whether they bind to additional protein targets in dividing cells. ne additional outcome of our results is the proposal of a double hit strategy for the same target protein, e.g. an exposure of tumor cells to a combination of ATP competitive and an ATP uncompetitive Eg5 targeting drugs as a possible treatment strategy to minimize or slow the development of drug resistance due to mutations in one of the drug binding sites. (1) Good JA, Skoufias DA, Kozielski F. Elucidating the functionality of kinesins with small molecule probes Seminars in Cell and Developmental Biology 22(9): (2) Tcherniuk S, R van Lis, F Kozielski, DA Skoufias Mutations in the human kinesin Eg5 that confer resistance to monastrol and S- Trityl- L- Cysteine in tumor derived cell lines. Biochemical Pharmacology 79(6): (3) Indorato RL, DeBonis S, Kozielski F, Garcia- Saez I, Skoufias DA. STLC- resistant cell lines as tools to classify chemically divergent Eg5 targeting agents according to their mode of action and target specificity Biochem Pharmacol Nov 15;86(10):

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